PROJECT ABSTRACT Cerebral cavernous malformations (CCMs), which can occur in 1 out of every 200 people in the US, cause seizure and cerebral hemorrhage. Previous studies have shown that decreased expression or a loss-of-function mutation of CCM family genes (CCM1, CCM2, CCM3) causes CCMs. Although the genetic origins of familial CCMs (20% of cases) have been well characterized, the etiology of sporadic CCMs (80% of cases) that do not carry a germline mutation in CCM genes remains to be established. The long-term goal of our research is to determine if and the extent to which Nogo-B receptor (NgBR) deficiency plays a causal role in the etiology of sporadic CCMs. Support for this idea comes from our previous R01-funded studies where we not only elucidated the molecular mechanisms by which NgBR binds farnesylated Ras to promote angiogenesis but also identified that the NgBR-mediated Ras pathway regulates the expression and activation of transcription factors in endothelial cells. Interestingly, these transcription factors have their binding elements in the promoter regions of CCM1 and CCM2 genes. Consequently, transcript levels of CCM1 and CCM2 genes decrease in NgBR-deficient human brain endothelial cells in vitro and in the yolk sac of NgBR endothelial cell-specific knockout (ecKO) mice in vivo. Additional support of the NgBR-CCM connection comes from histology studies showing that NgBR immunostaining intensity is significantly decreased in endothelial cells (ECs) of human sporadic CCM lesion tissue sections. NgBR ecKO mice have aberrant patterns of cerebral blood vessel assembly, which results in defective EC polarization and the formation of dilated blood vessels. This phenotype is surprisingly similar to the CCM lesions. In addition, the pericyte layer around blood vessels in NgBR ecKO mice is much thinner than littermate control mice. This means that endothelial NgBR deficiency can actually impair pericyte recruitment, which is known as essential for maintaining the structural integrity of blood vessels. Based on these findings, we hypothesize that the NgBR-Ras signaling pathway regulates CCM1/2 expression, and that disrupting this signaling pathway results in cerebrovascular malformation. We will test this hypothesis in three aims. Aim 1: Determine the role of the NgBR-CCM1/2 axis in regulating EC polarization and cerebral blood vessel assembly; Aim 2: Determine the role of the NgBR-CCM1/2 axis in maintaining pericyte recruitment and the structural integrity of cerebral blood vessels; Aim 3: Determine the mechanisms by which NgBR regulates CCM1/2 transcription and the contributions of NgBR-Ras pathway deficiency to the pathogenesis of sporadic CCM. The studies proposed here will establish clear links between NgBR, CCM1/2, and cerebrovascular malformation. Findings from our studies will provide new insight into how NgBR and NgBR-regulated factors guide CCM1/2 expression in brain ECs, and maintain the structural integrity of cerebral blood vessels.